AN INTERNALLY QUENCHED FLUOROGENIC SUBSTRATE OF PROHORMONE CONVERTASE-1 AND FURIN LEADS TO A POTENT PROHORMONE CONVERTASE INHIBITOR

Based upon the observed cleavage of various peptidyl substrates by the recombinant prohormone convertases PC1 and furin, an intramolecularly quenched fluorogenic peptidyl substrate, (o-aminobenzoyl)-Lys-Glu-Arg-Ser-Lys-Arg-Ser-Ala-Leu-Arg-Asp-(3-nitro)Tyr-Ala, was synthesized, In spite of the distance (approx. 33 Angstrom) separating the fluorescent donor/acceptor pair, the highly fluorescent o-aminobenzoyl group is, efficiently quenched by long-range resonance energy transfer to the (3-nitro)Tyr moiety. Both recombinant human PC1 and human furin recognize and cleave specifically this substrate at the expected Arg-Ser site in a sensitive manner. The K-m values for human PC1 and human furin were 17 mu M and 30 mu M respectively, with V-max values of 6.4 mu M/h and 18 mu M/h. These values differ significantly from those obtained when using a 7-amino-4-methylcoumarin-containing pentapeptidyl substrate where, for similar K-m values, the V-max. values were much lower. The peptide sequence was used to synthesize another peptide incorporating a ketomethylene arginyl pseudopeptide bond. This compound proved to be a potent competitive inhibitor of both human PC1 and human furin, displaying K-i values of 7.2 mu M and 2.4 mu M respectively.